Angina pectoris is chest pain or discomfort that is caused by insufficient blood flow to the heart muscle. The prevalence of angina in US adults over the age of 20 is estimated at 9,100,000. Stable angina pectoris is angina pectoris where the pain is predictable as occurring upon physical exertion or when the subject is under emotional stress. The prevalence of stable angina in US adults over the age of 45 (without corresponding myocardial infarction) is 500,000. See Rosamond et al. Circulation 117(4): e25 (2008).
Current therapies include aspirin, beta blockers (e.g., carvedilol, propranolol, atenolol), nitroglycerin (for acute relief), vasodilators such as calcium channel blockers (e.g., nifedipine (Adalat) and amlodipine), vsosorbide mononitrate and nicorandil, If channel inhibitors (e.g., ivabradine), ACE inhibitors, statins, and ranolazine (Ranexa). However, such therapies typically only treat the pain without preventing the pain from recurring and not all angina patients respond to such treatments. Clinical trials are being conducted with CD34 stem cells for treatment of refractory angina with the goal of using the stem cells to generate new vascularization to prevent the pain from recurring. However, this treatment has not yet been demonstrated to be safe and efficacious. Thus, there is a need for a long term treatment option for angina pectoris, particularly refractory angina pectoris.
The management of patients with refractory angina is complex and requires a multidisciplinary approach. Current therapies include aspirin, beta blockers (e.g., carvedilol, propranolol, atenolol), nitroglycerin (for acute relief), vasodilators such as calcium channel blockers (e.g., nifedipine (Adalat) and amlodipine), vsosorbide mononitrate and nicorandil, If channel inhibitors (e.g., ivabradine), ACE inhibitors, statins, and ranolazine (Ranexa). And such therapies typically involve exhaustive regimens of trying of numerous different drug regimens and combinations in an attempt to reduce the pain. However, such therapies typically only treat the pain without preventing the pain from recurring and not all angina patients respond to such treatments.
In addition to pharmaceutical therapies, angina may be treated with interventional procedures such as percutaneous transluminal coronary angioplasty or coronary artery bypass graft (CABG) surgery, but such therapies are unavailable to some angina patients due to any number of factors, such as unfavorable coronary anatomy bed, thin coronary arteries, distal or diffuse coronary lesions, etc. Recommendations in the American Heart Association 2002 Guidelines for alternative therapies include surgical laser transmyocardial revascularization (Class IIa), enhanced external counterpulsation and spinal cord stimulation (Class IIb).
New treatment modalities are under investigation including use of stem cell populations. A number of investigators have taken the first steps in demonstrating regeneration of heart tissue using bone marrow mononuclear stem cell containing fractions in the context of myocardial infarction. Such studies have observed: (i) Regeneration of myocardium after infarction; (ii) reduction of infarct size; and (iii) De novo expression of cardiac proteins by human bone marrow cells. Following up on these preliminary studies, several groups have demonstrated the regenerative potential of bone marrow-derived mesenchymal cells in different experimental heart models, with results primarily based upon their myogenic and angiogenic potential.
Clinical trials, mostly in acute myocardial infarction patients and with intracoronary stem cell delivery, have already been undertaken to examine the safety and efficacy of autologous cell transplantation for enhancement of cardiac repair. However, the trials have produced conflicting results without obvious basis in differences in study designs and cell populations or deliveries.
For ischemic heart disease, some clinical trials have demonstrated at least safety of bone marrow-derived mononuclear stem cells with variable degrees of efficacy. The most common delivery technique in these studies is intramyocardial infusion of stem cells—either transendocardial or transepicardial.
In the refractory angina setting, some groups have performed clinical trials mostly using bone marrow mononuclear cells (BMMCs), primarily as a sole treatment or in conjunction with CABG.
By way of example, Hamano et al. injected BMMCs through a transepicardial approach, in conjunction with CABG, in 5 “no option” patients with associated ischemic cardiomyopathy. Results showed an objective increase in myocardial perfusion in the injected area in three patients. However, this study is confounded by the effect of concomitant CABG, and the therapeutic effects of BMMCs treatment remain unclear.
Other investigators have reported their initial experience of endomyocardial injection of BMMCs, delivered with a percutaneous catheter as guided by electromechanical mapping (NOGA™ system). Overall, these nonrandomized studies have demonstrated that direct BMMCs transferred into the ischemic myocardium improved symptoms and exercise capacity and increased myocardial perfusion and function in patients with refractory angina in some but not all patients. Most of these studies enrolled “no option” patients with ischemic cardiomyopathy.
Recently, the first prospective randomized trial of BMMCs by endomyocardial injection in severe coronary disease (PROTECT-CAD) has been reported. This study showed a significant improvement in exercise time, left ventricular ejection fraction and stress-induced myocardial ischemia, in the treated group.
Losordo et al. performed a phase I/IIa, double-blind, randomized, placebo-controlled dose escalating clinical trial with endomyocardial injection of autologous CD34+ stem cells for refractory angina. Efficacy parameters including angina frequency, nitroglycerine usage, exercise time, and CCSAC class showed trends that favored CD34+ cell-treated patients versus control subjects given placebo.
Thus, for “no option” angina patients, clinical studies using bone marrow-derived mononuclear stem cells have shown some myocardial perfusion improvement and, to a lesser degree, improved ventricular function. Most of these studies included patients with ischemic cardiomyopathy, with moderate to severe left ventricular ejection fraction (LVEF) depression. However, to date no therapeutic based upon therapeutic cells (i.e., mononuclear cells or mesodermal stem cells) have been able to reliably reduce the pain or improve the perfusion in most or all patients treated.
Approximately 5 to 15% patients with chronic coronary artery disease present severe disabling angina pectoris which cannot be controlled by a combination of conventional therapy tools, including multiple series of drug therapy optimization treatments, percutaneous transluminal coronary angioplasty (PTCA), and coronary artery bypass grafting (CABG) (35, 37). Severe angina pectoris often results in a substantial decrease in quality of life. Symptom relief for the “no option” refractory angina patient is a complex and challenging process. Alternative therapies in compliance with the American Heart Association Guidelines (11, 14), such as surgical laser transmyocardial revascularization, external counterpulsation, and spinal cord stimulation have all provided modest results at best (9, 26, 31, 45). The vast majority of refractory angina pectoris patients (75%) have preserved left ventricular function, with a mortality rate lower than the general coronary heart disease population (36, 54); and this patient group is rapidly growing.
Cellular therapy, specifically autologous bone marrow cell transplantation, has emerged as a new therapeutic option for cardiac regeneration. Some hypothetical mechanistic explanations involve the stem cells' myogenic and angiogenic potential, and the activation of resident progenitor cell growth via paracrine effects (2, 15-17, 22, 48, 49, 52). Although regeneration of myocardial tissue and concomitant reduction of infarcted area have been demonstrated in experimental animal models, many uncertainties regarding the translation of these results into humans remain (7, 33), rendering BMMC transplantation for cardiac tissue regeneration an experimental procedure, not a standard of care for clinical practice.
On the other hand, refractory angina patients may benefit from cellular based therapy, particularly in regards to angiogenesis. These angiogenic effects are considered among investigators to be very important when cell therapy is considered an option for human patients (8).
Angiogenic effects were reported in several pre-clinical studies (8, 28, 33, 48). The improvement of angiogenesis was observed in hearts transplanted with c-kit bone marrow (BM) cells when compared with negative control mice (40). Mobilization of mouse BM cells into circulation after acute myocardial infarction resulted in regeneration of myocytes and vascular structures (39). A recent study in nonhuman primates using a similar protocol showed an improvement in local perfusion in the BM-treated animals, suggesting potential angiogenic effects (30). This functional benefit in BM cell implantation is likely attributed to a paracrine effect with an increase in angiogenesis through local release of multiple growth factors, such as vascular endothelial growth factor and stromal cell-derived factor-1, among others (8, 48).
In clinical studies involving refractory angina patients, who received bone marrow derived stem cells or BMMC, improvement in symptoms and exercise capacity, as well as in myocardial perfusion were observed (4, 6, 8, 12, 13, 19, 21, 41, 53, 55, 56, 59). Beeres et al. (3) conducted a trial using intramyocardial injection of autologous BMMC in 25 patients with refractory angina, which showed sustained beneficial effects on anginal symptoms and myocardial perfusion. Losordo et al. (34) performed a phase I/IIa, double-blind, randomized, placebo-controlled dose escalating clinical trial with endomyocardial injection of autologous CD34+ stem cells for refractory angina patients, demonstrating a trend of increased exercise time, in addition to Canadian Cardiovascular Society Angina Classification (CCSAC) improvement among CD34+ cell-treated patients. van Ramshorst et al. (58) conducted a randomized controlled trial of intramyocardial bone marrow cell injection for refractory angina, with a short-term follow up (3 to 6 months) showing a modest improvement in myocardial perfusion compared with placebo.
Observing the minimal left ventricular improvement and the clinical response of previous refractory angina trials, it is suggested that the primary action of bone marrow stem cell transplantation in humans is promoting myocardial angiogenesis, and not pure myogenesis. In this setting, angiogenesis can surely improve left ventricular function through rescuing or recruiting hibernating myocardium, but to a limited extent, as demonstrated by these trials and some meta-analysis (1, 32, 44).
Previous pre-clinical and clinical studies have supported the feasibility, safety, and efficacious potential of stem cell therapy for myocardium tissue regeneration and encompasses patients presenting with a range of diagnoses from acute myocardial infarction to chronic ischemic heart disease (33).
The greatest challenge here consists in translating laboratory results to the hospital routine. Differences in study design, stem and mononuclear cell preparation, and infusion techniques have delivered somewhat promising, but inconsistent overall data from different studies (43).
Cerebrovascular disease, considered one of the top five non-communicable diseases, affects approximately 50 million people worldwide, resulting in approximately 5.5 million deaths per year. Of those 50 million, stroke accounts for roughly 40 million people.
Like Angina, Stroke is another condition where ischemia plays a significant role. Stroke is the third leading cause of death in developed countries and accounts for the major cause of adult disability. Presently there is only one available treatment option. It is a vascular disease that impacts cognitive and motor function and alters the immune system. This study focused on the pathophysiology of stroke and the development of a novel cell therapy (human umbilical cord blood (HUCB) cells) that in early studies was shown to significantly improve motor dysfunction and reduce infarct size. The role of the immune/inflammatory response in the development of brain injury after stroke is not fully understood. After the ischemic event, there is an immune response resulting in the influx of neutrophils, T-cells, B-cells, natural killer cells as well as microglia into the infarcted hemisphere and a change in profile of these same immune cells in the peripheral blood. This study examined whether the beneficial effects of HUCB injection can be attributed to a specific cell population.
Stroke treatment consists of two categories: prevention and acute management. Prevention treatments currently consist of antiplatelet agents, anticoagulation agents, surgical therapy, angioplasty, lifestyle adjustments, and medical adjustments. An antiplatelet agent commonly used is aspirin. The use of anticoagulation agents seems to have no statistical significance. Surgical therapy appears to be effective for specific sub-groups. Angioplasty is still an experimental procedure with insufficient data for analysis. Lifestyle adjustments include quitting smoking, regular exercise, regulation of eating, limiting sodium intake, and moderating alcohol consumption. Medical adjustments include medications to lower blood pressure, lowering cholesterol, controlling diabetes, and controlling circulation problems.
Acute management treatments consist of the use of thrombolytics, neuroprotective agents, Oxygenated Fluorocarbon Nutrient Emulsion (OFNE) Therapy, Neuroperfusion, GPIIb/IIIa Platelet Inhibitor Therapy, and Rehabilitation/Physical Therapy.
A thrombolytic agent induces or moderates thrombolysis, and the most commonly used agent is tissue plasminogen activator (t-PA). Recombinant t-PA (rt-PA) helps reestablish cerebral circulation by dissolving (lysing) the clots that obstruct blood flow. It is an effective treatment, with an extremely short therapeutic window; it must be administered within 3 hours from onset. It also requires a CT scan prior to administration of the treatment, further reducing the amount of time available. Genetech Pharmaceuticals manufactures ACTIV ASE® and is currently the only source of rt-P A.
Neuroprotective agents are drugs that minimize the effects of the ischemic cascade, and include, for example, Glutamate Antagonists, Calcium Antagonists, Opiate Antagonists, GAB A-A Agonists, Calpain Inhibitors, Kinase Inhibitors, and Antioxidants. Several different clinical trials for acute ischemic stroke are in progress. Due to their complementary functions of clot-busting and brainprotection, future acute treatment procedures will most likely involve the combination of thrombolytic and neuroprotective therapies. However, like thrombolytics, most neuroprotectives need to be administered within 6 hours after a stroke to be effective.
Oxygenated Fluorocarbon Nutrient Emulsion (OFNE) Therapy delivers oxygen and nutrients to the brain through the cerebral spinal fluid. Neuroperfusion is an experimental procedure in which oxygen-rich blood is rerouted through the brain as a way to minimize the damage of an ischemic stroke. GPIIblIIIa Platelet Inhibitor Therapy inhibits the ability of the glycoprotein GPlIb/IIIa receptors on platelets to aggregate, or clump. Rehabilitational Physical Therapy must begin early after stroke, however, they cannot change the brain damage. The goal of rehabilitation is to improve function so that the stroke survivor can become as independent as possible.
Although some of the acute treatments showed promise in clinical trials, a study conducted in Cleveland showed that only 1.8% of patients presenting with stroke symptoms even received the t-PA treatment (Katz an IL, et ai., 2000 JAMA, 283:1151-1158). t-PA is currently the most widely used of the above-mentioned acute stroke treatments, however, the number of patients receiving any new “effective” acute stroke treatment is estimated to be under 10%. These statistics show a clear need for the availability of acute stroke treatment at greater than 24 hours post stroke.
For some of these acute treatments (Le., t-PA) the time of administration is crucial. Recent studies have found that 42% of stroke patients wait as long as 24 hours before arriving at the hospital, with the average time of arrival being 6 hours after stroke. t-PA has been shown to enhance recovery of −113 of the patients that receive the therapy, however a recent study mandated by the FDA (Standard Treatment with Alteplase to Reverse Stroke found that about a third of the time the three-hour treatment window was violated resulting in an ineffective treatment. With the exception of rehabilitation, the remaining acute treatments are still in clinical trials and are not widely available in the U.S., particularly in rural areas, which may not have large medical centers with the needed neurology specialists and emergency room staffing, access to any of these new methods of stroke diagnosis and therapy may be limited for some time.
The cost of stroke in the US is over $43 billion, including both direct and indirect costs. The direct costs account for about 60% of the total amount and include hospital stays, physicians' fees, and rehabilitation. These costs normally reach $15,000 patient in the first three months; however, in approximately 10% of the cases, the costs are in excess of $35,000. Indirect costs account for the remaining portion and include lost productivity of the stroke victim, and lost productivity of family member caregivers (see National Institute of Neurological Disorders and Stroke, NIH).
Approximately 750,000 strokes occur in the US every year, of which about ⅓ are fatal. Of the remaining patients, approximately ⅓ is impaired mildly, ⅓ is impaired moderately, and ⅓ is impaired severely. Ischemic stroke accounts for 80% of these strokes.
As the baby-boomers age, the total number of strokes is projected to increase substantially. The risk of stroke increases with age. After age 55, the risk of having a stroke doubles every decade, with approximately 40% of individuals in their 80's having strokes. Also, the risk of having a second stroke increases over time. The risk of having a second stroke is 25-40% five years after the first. With the over-65 portion of the population expected to increase as the baby boomers reach their golden years, the size of this market will grow substantially. Also, the demand for an effective treatment will increase dramatically.
Given the inability to effectively mitigate the devastating effects of stroke, it is imperative that novel therapeutic strategies are developed to both minimize the initial neural trauma as well as repair the damage brain once the pathological cascade of stroke has run its course.
Transplantation of monocytes has been proposed as a means of treating stroke. Because of the difficulty in effectively treating patients after stroke, there is a need in the art for methods to enhance the treatment of stroke.
Neovascularization is an integral process of inflammatory reactions and subsequent repair cascades in tissue injury. Monocytes/macrophages play key role in the inflammatory process including angiogenesis as well as the defense mechanisms by exerting microbicidal and immunomodulatory activity. Current studies have demonstrated that recruited monocytes/macrophages aid in regulating angiogenesis in ischemic tissue, tumors, and chronic inflammation. In terms of neovascularization followed by tissue regeneration, monocytes/macrophages should be highly attractive for cell-based therapy compared to any other stem cells due to their considerable advantages such as non-oncogenic, non-teratogenic, no ethical controversy, multiple secretary functions including pro-angiogenic and growth factors, and easy self-harvesting. Not only adult origins such as bone marrow or peripheral blood, but also umbilical cord blood (UCB) can be potential sources for autologous or allogeneic monocytes/macrophages. Especially, UCB monocytes should be considered as the first candidate owing to their fast feasibility, low immune rejection, and multiple skills such as anti-inflammatory reaction in virtue of unique immune and inflammatory immaturity as well as pro-angiogenic ability. General characteristics and potential of monocytes/macrophages are presented for cell-based therapy, especially focusing on neovascularization and UCB-derived monocytes.
One interesting function of monocytes/macrophages is to promote angiogenesis related to inflammatory reactions. Angiogenesis (or neovascularization) is a major element of inflammatory processes including subsequent repair cascades [Sunderkotter, 1994 #4]. During the early inflammatory process, circulating blood monocytes extravasate into tissues [Bosco, 2008 #3]. Initially, neighboring endothelial and inflammatory cells regulate this monocyte passing through vessel wall by releasing of a series of adhesion and chemotactic materials [Baggiolini, 2000 #9; Imhof, 2004 #2; Bosco, 2008 #3]. Along chemotactic and oxygen gradients between normal and injured tissues, extravasated monocytes move and gather into hypoxic and/or necrotic cores of diseased tissues before differentiation into tissue macrophages. The representative pathologic tissues to which monocytes/macrophages are apt to accumulate are as follows: solid tumors, myocardial or cerebral infarction, synovial joints of chronic arthritis or atheromatous plaques, bacterial infection, and healing wounds [Baggiolini, 2000 #9; Murdoch, 2004 #1; Bosco, 2008 #3; Mantovani, 2002 #15] (FIG. 1).
After differentiation from monocytes, macrophages in tissue have been known to exist as polarized populations, M1 and M2 subsets [Mantovani, 2004 #67; Sica, 2006 #16; Mantovani, 2004 #67; Mantovani, 2002 #15]. While M1 polarized macrophages are powerful inflammatory cells that produce pro-inflammatory cytokines and phagocytize pathogens, M2 macrophages modulate the inflammatory responses and help on angiogenesis and tissue repair [Mantovani, 2004 #67; Sica, 2006 #16; Mantovani, 2004 #67; Mantovani, 2002 #15]. Interestingly, in gene expression of macrophages, a combination of M1 and M2 subsets early in wound healing turns into dominantly M2 genes later [Deonarine, 2007 #68]. During the early stage of the wound healing process, M1 macrophages lead to an direct inflammatory reaction that clean up the wound and debris of microbes and/or injured host tissues whereas tissue repair and angiogenesis are begun by M2 macrophages at the same time. In the late stage when the cleansing by M1 macrophages is almost over, the prevailing M2 macrophages go on with their work, tissue regeneration including angiogenesis [Deonarine, 2007 #68]. Accumulating evidence suggests that recruited monocytes/macrophages aid in modulating and regulating neovascularization in ischemic tissue, tumors, and chronic inflammation such as arthritic joints and atherosclerosis.